Cis-acting Negative Regulatory Element Research Articles

Global transcriptional repression: An initial and essential step for Plasmodium sexual development

Gametocytes are nonreplicative sexual forms that mediate malaria transmission to a mosquito vector. They are generated from asexual blood-stage parasites that proliferate in the circulation. However, little is known about how this transition is genetically regulated. Here, we report that an Apetala2 (AP2) family transcription factor, AP2-G2, regulates this transition as a transcriptional repressor. Disruption of AP2-G2 in the rodent malaria parasite Plasmodium berghei did not prevent commitment to the sexual stage but did halt development before the appearance of sex-specific morphologies. ChIP-seq analysis revealed that AP2-G2 targeted ∼1,500 genes and recognized a five-base motif in their promoters. Most of these target genes are required for asexual proliferation of the parasites in the blood, suggesting that AP2-G2 blocks the program that precedes asexual replication to promote conversion to the sexual stage. Microarray analysis showed that the identified targets constituted ∼70% of the up-regulated genes in AP2-G2-depleted parasites, suggesting that AP2-G2 actually functions as a repressor in gametocytes. A promoter assay using a centromere plasmid demonstrated that the binding motif functions as a cis-acting negative regulatory element. These results suggest that global transcriptional repression, which occurs during the initial phase of gametocytogenesis, is an essential step in Plasmodium sexual development.

Positive and negative regulation in the promoter of the ORF46 gene of Kaposi's sarcoma-associated herpesvirus

The ORF46 gene of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes uracil DNA glycosylase, an enzyme involved in DNA repair. In this study, we show that the transcriptional start site of the ORF46 gene is located at nucleotide 69,425 of the viral genome and ORF50 protein, a latent-lytic switch transactivator, activates the ORF46 promoter via RBP-Jκ protein. Three consensus RBP-Jκ-binding sites found in the ORF46 promoter are critical for the binding of RBP-Jκ protein and conferring the ORF50 responsiveness. In addition, a negative regulatory region has been determined in the ORF46 promoter, which mediates the suppression of the ORF50 responsiveness. The functional negative region of the ORF46 promoter is mainly composed of the Sp1-binding sites. Like the negative region of the ORF46 promoter, addition of Sp1-binding sequences alone in an ORF50-responsive promoter efficiently confers the suppression of the ORF50 responsiveness. Furthermore, sodium butyrate, a pleiotropic inducing agent for the KSHV lytic cycle, is able to relieve the negative regulation of the ORF46 promoter in the latently KSHV-infected cells. The identification of multiple positive and negative cis-acting regulatory elements in the viral promoters emphasizes the elaborate controls in the KSHV lytic cycle, which ensure the adequate expression of each viral lytic gene.

Promoter Regulation of the Visinin-like Subfamily of Neuronal Calcium Sensor Proteins by Nuclear Respiratory Factor-1

VILIP-1 (gene name VSNL1), a member of the neuronal Ca(2+) sensor protein family, acts as a tumor suppressor gene by inhibiting cell proliferation, adhesion, and invasiveness. VILIP-1 expression is down-regulated in several types of human cancer. In human non-small cell lung cancer, we found that down-regulation was due to epigenetic changes. Consequently, in this study we analyzed the VSNL1 promoter and its regulation. Serial truncation of the proximal 2-kb VSNL1 promoter (VP-1998) from its 5' terminus disclosed that the last 3' terminal 100-bp promoter fragment maintained similar promoter activity as compared with VP-1998 and therefore was referred to as VSNL1 minimal promoter. When the 5' terminal 50 bp were deleted from the minimal promoter, the activity was dramatically decreased, suggesting that the deleted 50 bp contained a potential cis-acting element crucial for promoter activity. Deletion and site-directed mutagenesis combined with in silico transcription factor binding analysis of VSNL1 promoter identified nuclear respiratory factor (NRF)-1/alpha-PAL as a major player in regulating VSNL1 minimal promoter activity. The function of NRF-1 was further confirmed using dominant-negative NRF-1 overexpression and NRF-1 small interfering RNA knockdown. Electrophoretic mobility shift assay and chromatin immunoprecipitation provided evidence for direct NRF-1 binding to the VSNL1 promoter. Methylation of the NRF-1-binding site was found to be able to regulate VSNL1 promoter activity. Our results further indicated that NRF-1 could be a regulatory factor for gene expression of the other visinin-like subfamily members including HPCAL4, HPCAL1, HPCA, and NCALD.

Characterization of epididymal epithelial cell-specific gene promoters by in vivo electroporation.

The mammalian epididymis plays a critical role in sperm maturation, a function dependent on testicular androgens. However, the function of the initial segment, the most proximal part of the epididymis, is also dependent on luminal factors of testicular origin. Efferent duct ligation (EDL), which prevents luminal testicular fluid from reaching the epididymis, results in changes in gene expression within this region. Cystatin-related epididymal spermatogenic (cres) gene and gamma-glutamyl transpeptidase (GGT) mRNA IV are highly expressed in the initial segment and are regulated by luminal testicular factors. EDL results in decreased expression of both genes. To evaluate these promoters in the context of their native physiological state, an in vivo electroporation procedure was used. Significant differences were observed in vivo compared to previous in vitro results. Whereas two C/EBP sites were necessary for transcriptional activity from a 135-base-pair (bp) cres promoter in vitro, only the 5' site displayed functional activity in the in vivo system. A 135-bp GGT promoter IV construct was sufficient for reporter gene expression in vitro. However, in vivo, substantial expression was not observed until the construct was extended to 530 bp. Three polyoma enhancer activator 3 (PEA3) sites were found to be necessary for in vivo reporter gene expression from this construct. A cis-acting negative regulatory element between -530 and -681 bp was also identified that was not previously recognized in the in vitro studies. These studies demonstrate the utility of in vivo electroporation for elucidating promoter elements that may not be identified when traditional in vitro methods are used.

Identification of a negative response element in the human inducible nitric-oxide synthase (hiNOS) promoter: The role of NF-kappa B-repressing factor (NRF) in basal repression of the hiNOS gene.

Although nuclear factor (NF)-kappaB plays a central role in mediating cytokine-stimulated human inducible nitric-oxide synthase (hiNOS) gene transcription, very little is known about the factors involved in silencing of the hiNOS promoter. NF-kappaB-repressing factor (NRF) interacts with a specific negative regulatory element (NRE) to mediate transcriptional repression of certain NF-kappaB responsive genes. By sequence comparison with the IFN-beta and IL-8 promoters, we identified an NRE in the hiNOS promoter located at -6.7 kb upstream. In A549 and HeLa human cells, constitutive NRF mRNA expression is detected by RT-PCR. Gel shift assay showed constitutive NRF binding to the hiNOS NRE. Mutation of the -6.7-kb NRE site in the hiNOS promoter resulted in loss of NRF binding and increased basal but not cytokine-stimulated hiNOS transcription in promoter transfection experiments. Interestingly, overexpression of NRF suppressed both basal and cytokine-induced hiNOS promoter activity that depended on an intact cis-acting NRE motif. By using stably transformed HeLa cells with the tetracycline on/off expression system, reduction of cellular NRF by expressing antisense NRF increased basal iNOS promoter activity and resulted in constitutive iNOS mRNA expression. These data demonstrate that the transacting NRF protein is involved in constitutive silencing of the hiNOS gene by binding to a cis-acting NRE upstream in the hiNOS promoter.

Functional characterization of the 5′-flanking and the promoter region of the human UCP3 (hUCP3) gene

Uncoupling protein-3 (UCP3) is considered as an important regulator of energy expenditure and thermogenesis in humans. To get insight into the mechanisms regulating its expression we have cloned and characterized about 5 kb of the 5′-flanking region of the human UCP3 (hUCP3) gene. 5′-RACE analysis suggested a single transcription initiation site 187 bp upstream from the translational start site. The promoter region contains both TATA and CAAT boxes as well as consensus motifs for PPRE, TRE, CRE and muscle-specific factors like MyoD and MEF2 sites. Functional characterization of a 3 kb hUCP3 promoter fragment in multiple cell lines using a CAT-ELISA identified a cis-acting negative regulatory element between −2983 and −982 while the region between −982 and −284 showed greatly increased basal promoter activity suggesting the presence of a strong enhancer element. Promoter activity was particularly enhanced in the murine skeletal muscle cell line C 2C 12 reflecting the tissue-selective expression pattern of UCP3.

Spatial and Temporal Expression of a Polysphondylium Spore-Specific Gene

In the cellular slime mold Polysphondylium spherical masses of cells are periodically released from the base of the culminating sorogen. These whorls undergo a morphogenetic transformation from spherical to radial symmetry, marked by the early emergence of a radially symmetric prepattern on the whorl surface. In previous experiments, morphogenesis was followed by observing prestalk cell markers. Here we describe the isolation and characterization of a spore coat gene whose expression pattern is the negative image of the prestalk pattern. To study the molecular mechanism of sp-45 gene regulation, we have cloned and analyzed the sp-45 promoter. Deletion analysis localized a single positive regulatory element (PRE) to a 106-bp fragment between positions −246 and −352 of the upstream coding sequence. This fragment can be further divided into a promoter-proximal and promoter-distal PRE and a 29-bp sequence between them. The distal PRE can regulate prespore expression when fused to a nonfunctioning basal promoter. The distal PRE contains two adjacent essential elements, a Gr box (GTGATATAGTGG) and a TA box (TAATATATT). Each element can drive prespore cell-specific reporter gene expression independently when incorporated into a nonfunctional promoter. Our results also show that prespore cell-specific gene expression is solely under positive regulation, with no evidence for spore-specific enhancers or cis-acting negative regulatory elements. By fusing GFP to the C-terminus of sp-45, we have demonstrated that the graded gene expression of SP45 in the sorogen is regulated by a sequence lying within the sp-45 coding sequence. The temporal and spatial expression pattern of this protein, taken together with the prestalk expression pattern, demonstrates unambiguously that the radial symmetries that emerge in the whorl are established by a system of positional coordinates and that cell sorting plays little if any role in this process.

Functional Characterization of the HumanPAX3Gene Regulatory Region

Spatiotemporal expression of thePAX3gene is tightly regulated during development. We have isolated and sequenced the 5′-flanking regulatory region of humanPAX3.Primer extension and ribonuclease protection mapping revealed that transcription is initiated from a single start site downstream of a TATA-like motif in human brain and peripheral tissues. Functional dissection of the gene's 5′-flanking region, which had been fused to a luciferase reporter gene and transiently expressed in rhabdomyosarcoma (RD) and cos-7 cells, indicated that the upstream region ofPAX3contains multiple positive and negativecis-acting regulatory elements. While the basal promoter is likely to be driven by two CCAAT boxes located at nucleotide positions −90 and −135, a cluster of regulatory elements acting as a strong repressor was detected between nucleotides −1200 and −650. Comparison of human and murine sequences revealed more than 90% identity in this segment. A polymorphic (CA)nrepeat sequence and a G/C substitution are located 337 bp and 328 bp upstream of the transcription start site, respectively. PCR-based systematic screening for length variations in 225 unrelated individuals of a Caucasian population showed a bimodal distribution of multiple alleles containing between 13 and 30 repeat units. Although the (CA)25variant of thisPAX3gene-linked polymorphic region (PAX3LPR) conferred lower transcriptional efficiency on thePAX3promoter, a regulatory impact of the PAX3LPR on PAX3 expression related to brain plasticity and function remains to be demonstrated.

Functional analysis of the lysyl oxidase promoter in myofibroblast-like clones of 3T6 fibroblast.

Lysyl oxidase (LO), an extracellular enzyme catalysing the first step of collagen and elastin cross-linking, is transiently expressed by myofibroblasts during fibrosis. A cell model with features of myofibroblast was thus established for studying the regulation of LO. Two clones of the 3T6 fibroblast cell line were selected because 1) they produced a relatively high steady-state level of the three lysyl oxidase mRNAs with the same relative ratio similar to fibrotic tissue and 2) they stably displayed certain features of myofibroblast (alpha-smooth muscle actin cytoskeleton, bundles of cytoskeletal filaments beneath the cytoplasmic membranes). These clones synthesized predominantly type I collagen fibers and a small amount of type III collagen. Neither type IV collagen nor elastin were observed. The cloning and sequencing of 2,073 bp of the mouse Balb/C LO promoter was performed, allowing the identification around the initiation of transcription of consensus sequences which are found on the COL1 promoters. A series of deletion constructs containing the LO 5'-flanking region ligated to the luciferase gene were transiently transfected into 3T6-5 fibroblasts. The region allowing the maximal activity was found between positions -416 to -192, while the more upstream region negatively regulated the promoter. The -898 to -865 sequence (called LOcol1) displayed 79% of homology with a conserved sequence of murine, rat, and human COL1A1 promoters. This sequence participated to the binding of several nuclear factors within a region (-970 to -784) allowing 50% of inhibition of the LO promoter. Therefore, the level of LO transcription is regulated in 3T6-5 fibroblast by positive and negative cis-acting regulatory elements which might have common features with the COL1A1 promoter.

Functional analysis of the lysyl oxidase promoter in myofibroblast-like clones of 3T6 fibroblast

Lysyl oxidase (LO), an extracellular enzyme catalysing the first step of collagen and elastin cross-linking, is transiently expressed by myofibroblasts during fibrosis. A cell model with features of myofibroblast was thus established for studying the regulation of LO. Two clones of the 3T6 fibroblast cell line were selected because 1) they produced a relatively high steady-state level of the three lysyl oxidase mRNAs with the same relative ratio similar to fibrotic tissue and 2) they stably displayed certain features of myofibroblast (alpha-smooth muscle actin cytoskeleton, bundles of cytoskeletal filaments beneath the cytoplasmic membranes). These clones synthesized predominantly type I collagen fibers and a small amount of type III collagen. Neither type IV collagen nor elastin were observed. The cloning and sequencing of 2,073 bp of the mouse Balb/C LO promoter was performed, allowing the identification around the initiation of transcription of consensus sequences which are found on the COL1 promoters. A series of deletion constructs containing the LO 5'-flanking region ligated to the luciferase gene were transiently transfected into 3T6-5 fibroblasts. The region allowing the maximal activity was found between positions -416 to -192, while the more upstream region negatively regulated the promoter. The -898 to -865 sequence (called LOcol1) displayed 79% of homology with a conserved sequence of murine, rat, and human COL1A1 promoters. This sequence participated to the binding of several nuclear factors within a region (-970 to -784) allowing 50% of inhibition of the LO promoter. Therefore, the level of LO transcription is regulated in 3T6-5 fibroblast by positive and negative cis-acting regulatory elements which might have common features with the COL1A1 promoter.

Multiple nuclear regulatory proteins bind a single cis-acting promoter element to control basal transcription of the human alpha 4 integrin gene in corneal epithelial cells.

Expression of the fibronectin-binding integrin alpha 4 beta 1 has been postulated to be an important event in the process of corneal epithelial wound healing. In a previous study, we identified upstream positive and negative cis-acting regulatory elements that are needed to modulate the transcriptional activity of the human alpha 4 integrin subunit gene promoter in primary cultures of rabbit corneal epithelial cells. We have shown that most of the basal activity directed by this promoter was dependent on the presence of a cis-acting DNA sequence designated the alpha 4.1 element, centered at position -45 relative to the human alpha 4 mRNA start site. Here, we demonstrate that five distinct nuclear regulatory proteins (designated Bp1 to Bp5) from rabbit corneal epithelial cells possess the ability to bind the alpha 4.1 element in a specific manner in vitro. However, when they are combined together, only two of them (Bp2 and Bp5) retained their ability to interact with their specific target sequence in in vitro assays. The apparent molecular masses of the Bp1 to Bp5 proteins were determined and found to be of 91, 74, 59, 45, and 39 kD, respectively. Electrophoretic mobility-shift assays (EMSAs) indicated that only Bp2 also possesses the ability to bind the alpha 4.2 element, a site homologous to alpha 4.1 which plays a minor role in alpha 4 gene expression. Despite the presence of three Ets binding sites in the immediate vicinity of alpha 4.1, competition experiments in EMSA clearly indicate that Bp1, Bp2, Bp4, and Bp5 do not belong to the Ets family of transcription factors. Insertion of both alpha 4.1 and alpha 4.2 upstream from the basal promoter of the mouse p12 gene provided evidence that both elements have the ability to modulate basal expression driven from a heterologous promoter. alpha 4.1 was shown to function as an activator, whereas alpha 4.2 acted as a repressor in a manner that is dependent on its orientation, further stressing the critical regulatory function played by these two elements on alpha 4 gene basal expression.

Identification of a Novel Cis-acting Negative Regulatory Element Affecting Expression of the CYP1A1 Gene in Rat Epidermal Cells

Polycyclic aromatic hydrocarbons such as 3-methylcholanthrene are toxic to rat epidermal cells in low passages (3 to 6), but cultures of high passage (>/=15) are resistant. Since such compounds can be metabolically activated by cytochrome P4501A1, we have examined the regulation of this gene in low and high passage cells. Consistent with this difference, little or no 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible P4501A1 mRNA or enzyme activity was observed in high passage as compared to low passage cultures. Similarly, transfection of a luciferase reporter construct containing -1317 to +256 base pairs of the 5'-flanking region of the murine CYP1A1 gene was TCDD-inducible in low but not high passage cells. Ligand binding and transfection experiments demonstrated the presence of functional Ah receptor complexes in both high and low passage cells. Deletion analysis identified a 26-base pair negative regulatory DNA (NeRD) element contained within the upstream regulatory region of the CYP1A1 gene responsible for this effect. Nuclear extracts from both low and high passage cells contain a protein which specifically binds to NeRD-containing DNA. Thus, the loss of polycyclic aromatic hydrocarbon sensitivity in high passage rat epidermal cells appears to be due to decreased expression of CYP1A1, and this effect may be mediated by an altered NeRD binding factor(s) present in these cells.

Identification of nuclear and cytoplasmic proteins that interact specifically with an AU-rich, cis-acting inhibitory sequence in the 3' untranslated region of human papillomavirus type 1 late mRNAs

Expression of human papillomavirus late genes encoding L1 and L2 capsid proteins is restricted to terminally differentiated epithelial cells. We have previously identified and characterized an AU-rich, cis-acting negative regulatory element in the 3' untranslated region of human papillomavirus type 1 late mRNAs. This element acts posttranscriptionally to reduce mRNA levels and the translation efficiency of mRNAs. The experiments reported here are a continuation of our previous work. We have used RNA gel shifts and UV cross-linking assays to identify cellular proteins that interact with the inhibitory RNA sequence of human papillomavirus type 1. RNA gel shift assays established that cellular proteins interact with the AU-rich sequence. The binding of nuclear proteins was inhibited by competition with poly(U), whereas the binding of cytoplasmic proteins was inhibited by competition with poly(U) and also by competition with poly(A) and poly(G). Two nuclear proteins and two cytoplasmic proteins that bind specifically to the AU-rich RNA sequence were identified by UV cross-linking. These proteins did not bind to the 3' untranslated region of human papillomavirus type 1 early mRNAs, which does not show inhibitory activity. The cellular proteins identified in our experiments may therefore be involved in the inhibition of human papillomavirus type 1 late gene expression in nondifferentiated epithelial cells.

Cell-specific interaction of nuclear factors with a cis-acting negative regulatory element in the rat cardiac alpha myosin heavy chain gene

Cell-specific interaction of nuclear factors with a cis-acting negative regulatory element in the rat cardiac alpha myosin heavy chain gene

Binding of cellular repressor protein or the IE2 protein to a cis-acting negative regulatory element upstream of a human cytomegalovirus early promoter

We have previously shown that the human cytomegalovirus early UL4 promoter has upstream negative and positive cis-acting regulatory elements. In the absence of the upstream negative regulatory region, the positive element confers strong transcriptional activity. The positive element contains a CCAAT box dyad symmetry and binds the cellular transcription factor NF-Y. The effect of the negative regulatory element is negated by the viral IE2 protein (L. Huang, C.L. Malone, and M.F. Stinski, J. Virol. 68:2108, 1994). We investigated the binding of cellular or viral IE2 protein to the negative regulatory region. The major cis-acting negative regulatory element was located between -168 and -134 bp relative to the transcription start site. This element could be transferred to a heterologous promoter, and it functioned in either orientation. Mutational analysis demonstrated that a core DNA sequence in the cis-acting negative regulatory element, 5'-GTTTGGAATCGTT-3', was required for the binding of either a cellular repressor protein(s) or the viral IE2 protein. The cellular DNA binding activity was present in both nonpermissive HeLa and permissive human fibroblast cells but more abundant in HeLa cells. Binding of the cellular repressor protein to the upstream cis-acting negative regulatory element correlates with repression of transcription from the early UL4 promoter. Binding of the viral IE2 protein correlates with negation of the repressive effect.

Promoter region of the mouse cyclin-dependent kinase 5-encoding gene

While cyclin-dependent kinases, such as CDC2 and CDK2, are key regulators of cell-cycle progression, cyclin-dependent kinase 5 (CDK5) is highly expressed in mature neurons with no evident cell-cycle regulation. The 5′-region of the mouse CDK5 gene was isolated and sequenced. The isolated clone included exons 1 through 7. The 5′-flanking region has a high G+C content. There is no TATA box around the transcriptional start points ( tsp), as determined by primer extension analysis. One CCAAT box, one AP-1-binding site, two AP-2-binding sites, and one cAMP-responsive element are located upstream from the tsp. Promoter/ cat fusion assays showed that the 5.8-kb fragment of this 5′-flanking sequence possessed the promoter activities expressing cat in rat PC12 pheochromocytoma cells. The effect of deletions of the promoter suggested the presence of two negative control elements located from −2.9 kb to −546 bp, and from −212 to −155 upstream from the 5′ end of the tsp. Two positive elements from bp −300 to −212 and from −155 to −41 were also detected. In the element from bp −300 to −212, there was a putative NF-IL6-binding sequence. Thus, the CDK5 promoter region contains multiple positive and negative cis-acting regulatory elements, an arrangement which suggests that the regulation of transcription of CDK5 is under complex control.

In Vivo Modulation of the Rat Cytochrome P450 1A1 by Double-Stranded Phosphorothioate Oligodeoxynucleotides

CYP1A1 gene expression is regulated by known cis- and transacting elements controlling inhibition and induction of CYP1A1 transcription. The influence of a double-stranded phosphorothioate oligonucleotide (dsODN) with sequence identical to the CYP1A1 negative regulatory element (NRE) was examined in Sprague-Dawley rats. Two strategies were employed: (i) two single-stranded complementary 25-mer ODNs that form a double-stranded ODN (ODN1) and (ii) a 54-base, self-complementary ODN which forms a dsODN hairpin (ODN2). A dsODN hairpin with scrambled NRE sequence was evaluated as a control (ODN3). Zoxazolamine paralysis times, an in vivo marker of CYP1A1 activity, were reduced from 184 ± 18 min in saline-treated rats to 103 ± 12.5 min 24 hr after a single 1.7-mg ODN1 iv injection. Liver microsomal EROD, an in vitro marker of CYP1A1/2 activity, was increased from 210 ± 10 pmol in saline-treated animals to 703 ± 73 and 623 ± 89 pmol resorufin/mg protein/min after iv ODN1 and iv ODN2, respectively. ODN1′s activity did not change PNP hydroxylation and PROD, markers of CYPs 2E1 and 2B1/2. ODN2 did not significantly change PNP but did significantly alter PROD. The ODN3 did not cause any significant changes in any assay measured. The ODN1-induced responses in ZX paralysis and EROD were observed post-iv injection, but not following ip injection of ODN1. Western blot analysis of ODN1- and HPO-treated rat liver microsomes also revealed increased in CYP1A1 protein. These data indicate double-stranded ODNs mimic the cis-acting NRE in vivo inducing CYP1A1 in the absence of other xenobiotics.

Transcriptional regulation of the neuronal L-type calcium channel α1D subunit gene

1. The transcriptional regulation of the rat brain L-type calcium channel alpha 1D subunit (RB alpha 1D) gene was investigated using NG108-15 neuroblastoma-glioma cells. 2. Differentiation of NG108-15 cells in the presence of prostaglandin E1 or retinoic acid resulted in the appearance of mRNA encoding the RB alpha 1D subunit detected using Northern blot analysis. 3. A rat genomic DNA library was screened, and a 15.2-kb clone was isolated and partially sequenced which included part of the 5' upstream sequence through the initial part of intron 2 of the RB alpha 1D gene. 4. Deletion analysis, using a CAT reporter gene and transfected NG108-15 cells, revealed that the 1.2-kb 5'-upstream sequence from the RB alpha 1D gene contains cis-acting positive and negative regulatory elements. A deletion of the 3' end of exon 1 also suggested the presence of regulatory elements in the first exon. 5. DNase footprinting of exon 1 of the RB alpha 1D gene revealed two regions protected from digestion by specific protein binding, and the second region included an (ATG)7 trinucleotide repeat sequence. Electrophoretic mobility shift assays confirmed nuclear protein(s) binding to the (ATG)7 sequence. 6. The (ATG)7 sequence functions as a enhancer when linked to a thymidine kinase promoter and a CAT reporter gene. 7. These results provide the initial description of the transcriptional regulation of the RB alpha 1D gene and identify a novel enhancer that consists of an (ATG)7 trinucleotide repeat sequence.

Functional analysis of human alpha 1(I) procollagen gene promoter. Differential activity in collagen-producing and -nonproducing cells and response to transforming growth factor beta 1.

To gain a further understanding of the regulation of human type I collagen gene expression under physiologic and pathologic conditions, we characterized 5.3 kilobase pairs (kb) of the human alpha 1(I) procollagen gene promoter. A series of deletion constructs containing portions of the alpha 1(I) procollagen 5'-flanking region (with end points from -5.3 kb to -84 base pairs (bp)) ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into NIH/3T3 cells. Maximal CAT activity was observed with constructs having 5' end points from -804 to -174 bp. A further 5' deletion to -84 bp caused a marked reduction in CAT activity. Cells transfected with plasmids containing longer 5'-flanking fragments of the alpha 1(I) procollagen gene (-2.3 or -5.3 kb) showed reduced CAT activity compared with the -804 bp construct. The activity of the alpha 1(I) procollagen promoter was much lower in cells that do not normally express type I collagen (HeLa cells) compared with collagen-producing NIH/3T3 cells. The CAT activity of deletion constructs containing longer 5' regions than -84 bp was increased by approximately 2-fold in NIH/3T3 cells treated with transforming growth factor beta 1 (TGF beta 1). Electrophoretic mobility shift assays indicated that protein-DNA complex formation with a probe corresponding to the -170 to -80 bp fragment of the alpha 1(I) procollagen promoter was markedly enhanced in nuclear extracts prepared from TGF beta 1-treated fibroblasts as compared with untreated fibroblasts. The DNA binding activity stimulated by TGF beta 1 was specific for an Sp1-like sequence at positions -164 to -142 bp in the promoter. These results demonstrate that 1) there are both positive and negative cis-acting regulatory elements in the human alpha 1(I) procollagen promoter, 2) these regulatory regions function differently in collagen-producing and -nonproducing cells, 3) the alpha 1(I) procollagen promoter contains TGF beta 1-responsive sequences located between -174 and -84 bp from the transcription start site, and 4) TGF beta 1 caused marked stimulation of the DNA binding activity of a nuclear factor interacting with an Sp1-like binding site located within a region encompassing -164 to -142 bp of the alpha 1(I) procollagen promoter.

The 30-kDa rat liver transcription factor nuclear factor 1 binds the rat growth-hormone proximal silencer.

Transcription of the gene encoding rat growth hormone is under the influence of cis-acting negative regulatory elements termed silencers. We showed previously that one such element, designated the rat growth hormone proximal silencer-1 site, binds a nuclear protein, the nuclear-factor-1-like protein that is probably a member of the CAAT transcription factor/nuclear-factor-1 (CTF/NF-I) family of transcription factors. This nuclear protein possesses DNA-binding activity as well as biochemical properties similar to those reported for the 30-kDa rat liver form of nuclear factor 1 (NF1-L). Results from both gel mobility supershift assays and Western-blot analyses, performed in combination with a polyclonal antibody directed against the DNA-binding domain of NF1-L, indicated that rat liver nuclear factor 1 might indeed correspond to one of the transcription factors interacting with the rat growth-hormone proximal silencer element. Further experiments using gel mobility shift assays also indicated that, as for NF1-L, multiple proteins among the 52-66-kDa CTF/NF-I isoforms from human HeLa cells also possess the ability to bind the rat growth-hormone silencer.